Non-surgical, localized delivery of compositions for placental growth factors

ABSTRACT

Described herewith are compositions comprising placental growth factors and methods for non-surgical, localized delivery thereof. The composition is delivered to a diseased or injured organ and/or body part and is formulated in a manner which allows for localized retention of the composition at the site of delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/845,199, filed on Sep. 3, 2015, which is a continuation-in-part ofU.S. application Ser. No. 14/157,444, filed on Jan. 16, 2014, now U.S.Pat. No. 9,655,948, issued May 23, 2017, which claims the benefit ofU.S. Provisional Application Ser. No. 61/956,185, which was convertedfrom U.S. application Ser. No. 13/744,331, filed Jan. 17, 2013; all ofwhich are incorporated hereby by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention is directed, in part, to compositions comprisingplacental growth factors and methods for non-surgical, localizeddelivery thereof. In one embodiment, the composition is delivered to adiseased or injured organ and/or body part and is co-delivered with anagent which allows for localized retention of the composition at thesite of delivery.

State of the Art

Heretofore, modified placental tissue has been used to treat a diseasedor injured internal organ or body part. However, such use has beenlimited by the amount of tissue available and the size of the organ. Asa general rule, the minimum amount of modified placental tissue toelicit the desired result has been used. For example, in one embodiment,the placental tissue is used as a barrier layer between organs so as toprevent adhesion formation. See, for example, U.S. Patent ApplicationPublication No. 2010/0104539.

The in vivo placement of a modified placental tissue also requires aninvasive process whereby the placement requires an incision whichtypically accompanies surgery. However, injectable solutions containinga suspension of placental tissue recently have been used to provide fornon-invasive delivery of the placental tissue. While this approachallows for direct delivery of the placental tissue to the in vivodelivery site, the size and amount of the placental tissue so deliveredis limited by the width of the injection needle and the volumedelivered.

Therefore the use of micronized placental tissue particles in aninjectable form has provided significant benefits to disease or injuredtissue. However, notwithstanding their commercial success, the retentiontime of such compositions in vivo is limited by virtue of their verylarge surface area arising from such micronized particles. The deliveryor placement of compositions having less surface area than micronizedparticles, but more surface area than the placental tissue grafts fromwhich they are formed, would provide for a more sustained benefit to thepatient.

SUMMARY OF THE INVENTION

This invention is based, in part, on the discovery that a bioerodible orbiodegradable mass formed from placental tissue allows for extendedrelease of growth factors from the mass over a prolonged period of time.

This invention is also based on the discovery that localized delivery ofthe composition can be achieved by co-delivery in the solution orsuspension of an agent which allows for localized retention of thesolution or suspension at the site of delivery. Such agents includethixotropic agents, phase changing agents, and the like. Whenco-delivered, these agents form a viscous or gel-like bioerodible orbiodegradable mass in vivo which limits transport away from the site ofdelivery and allows for the diffusion of the growth factors from themass formed over a period of time.

Accordingly, in one aspect of this invention there is provided acomposition comprising a defined surface area formed from micronizedplacental tissue. a sufficient amount of placental growth factor totreat a diseased or injured organ and a body part wherein saidcomposition forms a localized mass when applied to or proximate to saiddiseased or injured organ or body part

In another aspect of this invention, the composition contains modifiedplacental tissue particles as defined herein. In another embodiment, thecomposition is free of modified placental tissue particles.

In another aspect, there is provided a method for preparing acomposition for localized delivery of placental growth factors, whichmethod comprises forming a porous bioerodible or biodegradable massformed from micronized placental tissue. In another embodiment, theporous mass can be formed in situ by combining an aqueous suspension ofplacental tissue particles or an aqueous solution of placental growthfactors with a sufficient amount of an agent which allows for localizedretention of the solution or suspension at the site of delivery. Suchagents include thixotropic agents, phase changing agents, and the like.

The biocompatible thixotropic agent is selected, by way of example only,from hyaluronic acid, collagen, thrombin gels, fibrin gels and fibringlues. In another embodiment, the phase changing agent is a gel formingagent, such as a Pluronic® (e.g., a copolymer of oxyethylene andoxypropylene). Preferably, any polymer used as a thixotropic agent or aphase changing agent is bioerodible. In yet another embodiment, the bodypart is selected from the group consisting of skin, mucosal membrane,gum adjacent to teeth, bone, cartilage, tendon, retina, peripheralnerve, peripheral nerve sheath, small intestine, large intestine,stomach, skeletal muscle, heart, liver, lung, and kidney.

A thixotropic composition is one where in the absence of shear, thecomposition has infinite viscosity (it does not move) and in thepresence of shear, the composition's viscosity is greatly reduced so asto be deliverable under shear. An example of a thixotropic compositionis toothpaste. A phase-changing composition is an aqueous compositionwhich undergoes a change from a liquid to a gel or solid mass based on asuitable trigger such as an increase in temperature, light activation,electromagnetic stimulation, the addition of a phase-changing co-factor(e.g., alginates plus calcium). Such compositions are well known in theart. These compositions are preferably deliverable under injection butalso can be delivered topically as necessary. If the viscosity of thecomposition does not permit conventional injection, high pressuresyringes can be used and are well known in the art. Non-limitingexamples of such high pressure syringes include those described in U.S.Pat. No. 6,503,244 (incorporated herein by reference in its entirety)and the like.

In one aspect of this invention there are provided, compositionscomprising a sufficient amount of placental growth factors to treat adiseased or injured organ and a body part wherein said composition is inthe form of a localized bioerodible mass when applied to or proximate tosaid diseased or injured organ or body part, wherein said compositionfurther comprises micronized placental tissue particles capable of beingfiltered through a sieve having a pore size from about 500 μm to about10 μm.

In one aspect of this invention there are provided, methods forpreparing a composition for localized delivery of placental growthfactors, comprising combining an aqueous suspension of micronizedplacental tissue particles, the micronized placental tissue particlesare capable of being filtered through a sieve having a pore size fromabout 500 μm to about 10 μm, an aqueous solution of placental growthfactors, and a sufficient amount of a localization agent, whereby thecomposition is locally retained at the site of delivery uponadministration.

In some embodiments, the composition further comprises a localizationagent, for example, a thixotropic agent, or a phase changing agent. Insome embodiments, the thixotropic agent is selected from the groupconsisting of hyaluronic acid, collagen, thrombin gels, fibrin gels andfibrin glues. In other embodiments, the phase changing agent is selectedfrom the group consisting of a gel forming agent, for example, acopolymer or tripolymer of oxyethylene and oxypropylene units. In yetother embodiments, the localization agent is selected from the groupconsisting of a hydrogel, a polymer, and a collagen gel.

In some embodiments, the micronized placental tissue particles capableof being filtered through a sieve having a pore size from about 300 μmto about 10 μm, or from about 250 μm to about 25 μm.

DETAILED DESCRIPTION OF THE INVENTION

Before this invention is disclosed and described, it is to be understoodthat the aspects described below are not limited to specificcompositions, synthetic methods, or uses as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting.

This invention is predicated in part on the discovery that the localizeddelivery of growth factors can be achieved either using a solution ofsuch growth factors or a suspension of a sufficient amount of modifiedplacental tissue combination with an agent that imparts a sufficientlevel of solidification in vivo so as to provide for a depot of growthfactors to treat a diseased or injured body part.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” or “patient” as used herein refers to any vertebrateorganism including, but not limited to, mammalian subjects such ashumans, farm animals, domesticated pets and the like.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

The term “exterior surface” refers to either or both surfaces of themodified placental tissue which will contact the organ of the patient towhich tissue is applied.

The term “organ” as used herein is used to have an ordinary meaning inthe art, and refers to organs constituting animal viscera in general.

The term “diseased” as used herein refers to an organ and/or body partthat is characterized as being in a disease state, or susceptible tobeing in a disease state, wherein the disease is amenable to treatmentwith placental growth factors.

The term “injured” as used herein is used to have an ordinary meaning inthe art, and includes any and all types of damage to an organ and/orbody part, wherein the injury is amenable to treatment with placentalgrowth factors.

The term “biocompatible” as used herein refers to a material that issuitable for implantation or injection into a subject. In variousaspects, a biocompatible material does not cause toxic or injuriouseffects once implanted in the subject.

The term “modified placental tissue” refers to any and all components ofplacental tissue including whole placental tissue that has been modifiedby cleaning, disinfecting, and/or segmenting the tissue as well as toseparated components of placental tissue such as amnion, chorion, theumbilical cord, and the like. Modified tissue may maintain cellularlayers, such as the epithelial layer and/or the fibroblast layer.Modified placental tissue may include further modification, such aslamination of one or more layers of placental tissue, micronization ofplacental tissue, chemisorption or physisorption of small molecules,proteins (e.g. growth factors, antibodies), nucleic acids (e.g.aptamers), polymers, or other substances.

The term “modified placental tissue particles” refers to modifiedplacental tissue particles which have been made into particles smallenough to form a suspension suitable for injection through a syringe.Such particles are preferably no more than about 300 microns in size,preferably less than about 250 microns, less than about 200 microns,less than about 150 microns, less than about 100 microns, or less thanabout 50 microns.

The term “placental growth factors” refers to that array of growthfactors obtainable from modified placental tissue. The manner ofobtaining such growth factors is not critical to the invention andinclude, by way of example only, aqueous extraction from the placenta,culturing of placental cells expressing such growth factors, and thelike. The concentration of extracted growth factors can be increased byreducing the volume of water, saline, or buffer used to extract thegrowth factors, by addition of growth factors produced from placentalcell cultures, and the like.

The term “sufficient amount” or “therapeutic amount” refers to an amountof placental growth factors that is sufficient to treat an injured ordiseased organ or body part. The “sufficient amount” will vary dependingon a variety of factors, such as but not limited to, the type and/oramount of placental tissue used, the type and/or size of the intendedorgan and/or body part to be treated, the severity of the disease orinjury to the organ and/or body part to be treated and theadministration route. The determination of a “sufficient amount” can bemade by one of ordinary skill in the art based on the disclosureprovided herein.

The term “proximate to” as used herein means adjacent to, or on a bodypart such that the placental growth factors exert the desired effect. Ingeneral, “proximate to” means a distance that is generally within theskill of the art but preferably is within about 3 cm, about 2 cm, about1 cm of, or on or in the organ or body part.

As used herein, the term “bioerodible,” which is used hereininterchangeably with the term “biodegradable,” refers to a biocompatiblematerial that gradually decomposes, dissolves, hydrolyzes and/or erodesin situ, or that is susceptible to degradation into smaller componentsor molecules in a living organism over a prolonged period of time, forexample, over days or months, such that the material is harmless to theliving organism under normal living conditions. Generally, the“bioerodible” polymers herein are polymers that are hydrolyzable, andbioerode in situ primarily through hydrolysis. Preferably, the smallercomponents or molecules are biocompatible to a patient.

As one of ordinary skill in the art would understand, the degradation ofthe material results in a continuous release of a therapeutic amount ofplacental growth factors incorporated in the material over a prolongedperiod of time, such as about 3 days, about 5 days, about 10 days, about15 days, about 20 days, about 25 days, about 30 days, about 2 months,about 3 months, about 4 months, about 5 months, or about 6 months. Adesired release rate can be determined and/or achieved by adjusting theinitial concentration of the growth factors incorporated in thebioerodible or biodegradable mass and the degradation rate of the mass.

A bioerodible or biodegradable mass has the benefit of localizedretention at or proximate to the site of injection. In addition,placement of the bioerodible or biodegradable mass is configured toretain a substantial portion of the surface area found in the micronizedplacenta tissue components which form the mass. For example, the masscan be configured into a honeycomb shape whereby the pores provide forsignificant surface area retention. A mold containing a plurality ofspikes, teeth, prongs, or the like in both the male and female moldhalves will result in a porous bioerodible or biodegradable mass havingsignificant amounts of surface area. Additional surface area can becreated by laser drilling into the mass. Other means to form a porousmass are well known in the art. As discussed above, a bioerodible orbiodegradable mass retains a substantial portion of its surface area.For example, a bioerodible or biodegradable mass may retain about 5%,about 10%, about 20%, about 30%, about 40%, or about 50% of its surfacearea for a period of time such as, for example, about 3 days, about 5days, about 10 days, about 15 days, about 20 days, about 25 days, about30 days, about 2 months, about 3 months, about 4 months, about 5 months,or about 6 months. In all cases such is significantly greater than aplacental tissue graft. Of course the degradation rate will depend uponthe amount of surface area exposed in vivo, whereby the greater theexposed surface area the faster the rate of degradation.

The period for sustained release of growth factors from the bioerodibleor biodegradable mass relates to the surface area exposed tophysiological fluid when said mass is injected or implanted in vivo.Based on the information provided herein, the skilled artisan canroutinely assess the duration of sustained release based directly, atleast in part, on the size of the mass and surface area of the mass.Using such parameters, the skilled artisan can form a suitable mass witha predetermined period of sustained release by simple correlations.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

In one embodiment, placental tissue may be modified as described in U.S.Provisional Application Ser. No. 61/683,698, including cleaning,separation of the amnion and chorion, removal or maintenance of theepithelial cell layer, decontamination, and dehydration. Dehydration maybe accomplished using the drying apparatus as described in U.S.Provisional Application Ser. No. 61/683,698. Both of which applicationsare incorporated herein by reference in their entireties. Each aspect ofthat process produces modified placental tissue for the purposes of thisinvention whether used alone or in combination. However, it is preferredthat the modification of placental tissue includes at least the steps ofcleaning and decontamination. As such, modified placental tissuepreferably comprises placental tissue which has been cleaned anddecontaminated and also includes placental tissue which has undergoneone or more of separation of the amnion and chorion, removal of theepithelial cell layer, and dehydration.

In some embodiments of the present technology, the modified placentaltissue is selected from amnion, chorion, or both amnion and chorion. Inexemplary embodiments, modified placental tissue does not include theumbilical cord.

Modified placental tissue can also be formed into layers which may bedried separately and laminated together or dried together to formmulti-layer laminates.

Described herein are compositions composed of micronized placentalcomponents and pharmaceutical compositions thereof. In one aspect, thecomposition includes (a) micronized amnion, chorion, intermediate tissuelayer, or any combination thereof and (b) a pharmaceutically acceptablecarrier. In one aspect, the composition includes micronized amnion andintermediate tissue layer. In another aspect, the composition includesmicronized amnion and chorion. Micronized placental tissue may besandwiched between one or more layers of a multilayer laminate, or ontop of a laminate. Micronized placental tissue may also be added to asingle layer of modified placental tissue. See, for example, U.S.Provisional Application Ser. No. 61/683,700, which is incorporatedherein by reference in its entirety.

Once the amnion, chorion, and/or intermediate tissue layer have beendehydrated individually or in the form of a tissue graft, the dehydratedtissue(s) is micronized. The micronized compositions can be producedusing instruments known in the art. For example, the Retsch OscillatingMill MM400 can be used to produce the micronized compositions describedherein. The particle size of the materials in the micronized compositioncan vary as well depending upon the application of the micronizedcomposition. In one aspect, the micronized composition has particlesthat are less than 500 μm, less than 400 μm, less than 300 μm, or from25 μm to 300 μm, from 25 μm to 200 μm, or from 25 μm to 150 μm. Incertain aspects, particles having a larger diameter (e.g. 150 μm to 350μm) are desirable.

Any method known by one of skill in the art may be used to separateparticles by size including by, for example, centrifugation,sedimentation, and sieve techniques. In one embodiment, sieves are usedto separate particles by size. The sieve size (i.e., the sieve poreopening) is about 500 μm to about 10 μm, about 400 μm to about 20 μm,about 350 μm to about 25 μm, about 300 μm to about 35 μm, about 300 μmto about 45 μm, about 250 μm to about 50 μm, about 210 μm to about 60μm, about 175 μm to about 75 μm, about 150 μm to about 80 μm, about 125μm to about 100 μm, or any range therein. In some embodiments the sievesize (e.g., opening) is about 500 μm, about 400 μm, about 350 μm, about300 μm, about 250 μm, about 210 μm, about 175 μm, about 150 μm, about125 μm, about 100 μm, about 80 μm, about 75 μm, about 60 μm, about 50μm, about 45 μm, about 35 μm, about 25 μm, about 20 μm, about 10 μm.

One of skill in the art will appreciate that when particles areseparated using a sieve, a dimension of at least a portion of theparticles may be larger than the opening of the sieve used. Said anotherway, when the shape of a particle resembles that more of a rod, thelonger axis may be about 25% longer than the shorter axis and thereforea particle having a shorter axis of under 75 μm but with a longer axisof about 100 μm may still be collected using a 75 μm sieve. In someembodiments, the longer axis of a micronized placental particle may beabout 30% longer than the shorter axis, about 25% longer than theshorter axis, about 20% longer than the shorter axis, about 15% longerthan the shorter axis, about 10% longer than the shorter axis, about 5%longer than the shorter axis, or about 1% longer than the shorter axis.

In one aspect, micronization is performed by mechanical grinding orshredding. In another aspect, micronization is performed cryogenicgrinding. In this aspect, the grinding jar containing the tissue iscontinually cooled with liquid nitrogen from the integrated coolingsystem before and during the grinding process. Thus the sample isembrittled and volatile components are preserved. Moreover, thedenaturing of proteins in the amnion, intermediate tissue layer, and/orchorion is minimized or prevented. In one aspect, the CryoMillmanufactured by Retsch can be used in this aspect.

The selection of components used to make the micronized componentsdescribed herein can vary depending upon the end-use of the composition.For example, amnion, chorion, intermediate tissue layer, or anycombination thereof as individual components can be admixed with oneanother and subsequently micronized. In another aspect, one or moretissue grafts composed of one or more amnion, chorion, intermediatetissue layers, or any combination thereof (i.e., laminates) can bemicronized. In a further aspect, one or more tissue grafts composed ofone or more amnion, chorion, intermediate tissue layers, or anycombination can be admixed with amnion, chorion, intermediate tissuelayer, or any combination thereof as individual components andsubsequently micronized.

The amount of different components used to make the micronizedcompositions described herein can vary depending upon the application ofthe micronized composition. In one aspect, when the micronizedcomposition is composed of amnion (with or without the intermediatetissue layer) and intermediate tissue layer, the weight ratio of amnionto intermediate tissue layer is from 10:1 to 1:10, 9:1 to 1:1, 8:1 to1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to1:1, or about 1:1. In another aspect, when the micronized composition iscomposed of amnion (with or without the intermediate tissue layer) andchorion, the weight ratio of chorion to amnion is from 10:1 to 1:10, 9:1to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1to 1:1, 2:1 to 1:1, or about 1:1.

In addition to amnion, the intermediate tissue layer, and chorion,additional components can be added to the composition prior to and/orafter micronization. In one aspect, a filler can be added. Examples offillers include, but are not limited to, allograft pericardium,allograft acellular dermis, Wharton's jelly separated from vascularstructures (i.e., umbilical vein and artery) and surrounding membrane,purified xenograft Type-1 collagen, biocellulose polymers or copolymers,biocompatible synthetic polymer or copolymer films, purified smallintestinal submucosa, bladder acellular matrix, cadaveric fascia, or anycombination thereof.

In another aspect, a bioactive agent can be added to the compositionprior to and/or after micronization. Examples of bioactive agentsinclude, but are not limited to, naturally occurring growth factorssourced from platelet concentrates, either using autologous bloodcollection and separation products, or platelet concentrates sourcedfrom expired banked blood; bone marrow aspirate; stem cells derived fromconcentrated human placental cord blood stem cells, concentratedamniotic fluid stem cells or stem cells grown in a bioreactor; orantibiotics. Upon application of the micronized composition withbioactive agent to the region of interest, the bioactive agent isdelivered to the region over time. Thus, the micronized particlesdescribed herein are useful as delivery devices of bioactive agents andother pharmaceutical agents when administered to a subject. Releaseprofiles can be modified based on, among other things, the selection ofthe components used to make the micronized compositions as well as thesize of the particles.

In a further aspect, the amnion can be cross-linked with theintermediate tissue layer, chorion, or a second amnion tissue. Forexample, a cross-linking agent can be added to the composition (e.g.,amnion, chorion, intermediate tissue layer, or any combination thereofas individual components and/or as tissue grafts) prior to and/or aftermicronization. In general, the cross-linking agent is nontoxic andnon-immunogenic. When the amnion, intermediate tissue layer, and/orchorion (or a tissue graft thereof) are treated with the cross-linkingagent, the cross-linking agent can be the same or different. In oneaspect, the amnion, intermediate tissue layer, and chorion can betreated separately with a cross-linking agent or, in the alternative,the amnion, intermediate tissue layer, and chorion can be treatedtogether with the same cross-linking agent. In certain aspects, theamnion, intermediate tissue layer, and chorion can be treated with twoor more different cross-linking agents. The conditions for treating theamnion, intermediate tissue layer, and chorion can vary. In otheraspects, the amnion, intermediate tissue layer, and/or chorion can bemicronized, and the micronized composition can subsequently be treatedwith a cross-linking agent. In one aspect, the concentration of thecross-linking agent is from 0.1 M to 5 M, 0.1 M to 4 M, 0.1 M to 3 M,0.1 M to 2 M, or 0.1 M to 1 M.

The cross-linking agent generally possesses two or more functionalgroups capable of reacting with proteins to produce covalent bonds. Inone aspect, the cross-linking agent possesses groups that can react withamino groups present on the protein. Examples of such functional groupsinclude, but are not limited to, hydroxyl groups, substituted orunsubstituted amino groups, carboxyl groups, and aldehyde groups. In oneaspect, the cross-linker can be a dialdehyde such as, for example,glutaraldehyde. In another aspect, the cross-linker can be acarbodiimide such as, for example,(N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC). In otheraspects, the cross-linker can be an oxidized dextran, p-azidobenzoylhydrazide, N-[alpha-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[beta-(4-azidosalicylamido)ethyl]disulfide,bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate,disuccinimidyl suberate, and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, abifunctional oxirane (OXR), or ethylene glycol diglycidyl ether (EGDE).

In one aspect, sugar is the cross-linking agent, where the sugar canreact with proteins present in the amnion, intermediate tissue layer,and chorion to form a covalent bond. For example, the sugar can reactwith proteins by the Maillard reaction, which is initiated by thenonenzymatic glycosylation of amino groups on proteins by reducingsugars and leads to the subsequent formation of covalent bonds. Examplesof sugars useful as a cross-linking agent include, but are not limitedto, D-ribose, glycerone, altrose, talose, ertheose, glucose, lyxose,mannose, xylose, gulose, arabinose, idose, allose, galactose, maltose,lactose, sucrose, cellibiose, gentibiose, melibiose, turanose,trehalose, isomaltose, or any combination thereof.

In certain aspects, the micronized composition can be used to form athree-dimensional construct. For example, the micronized particles canbe treated with a cross-linking agent described above then placed in amold having specific dimensions. Alternatively, the micronized particlescan be placed into the mold and subsequently treated with thecross-linking agent. In one aspect, the cross-linked particles can bemanually formed into any desired shape. In other aspects, one or moreadhesives can be admixed with an adhesive prior to being introduced intothe mold. Examples of such adhesives include, but are not limited to,fibrin sealants, cyanoacrylates, gelatin and thrombin products,polyethylene glycol polymer, albumin, and glutaraldehyde products. Notwishing to be bound by theory, the three-dimensional construct composedof smaller micronized particles will produce a denser product capable ofbearing mechanical loads. Alternatively, larger micronized particleswill produce constructs that are less dense and possess compressiveproperties. This feature can be useful in non-load void filling,especially where it is desirable to have a product that will conform toirregular shapes. The three-dimensional constructs can include one ormore bioactive agents described herein.

A bioerodible or biodegradable mass formed by the molds described abovecan have an added benefit of an increased surface area. Additionally, amold can create a number of indentations, undulations, or the like onthe surface and/or within the mass such that the surface area is furtherincreased. Decreasing the surface area of the bioerodible orbiodegradable mass allows for a slower rate of degradation such that themass may take, for example, two, three, four, five, or more times aslong to decompose, dissolve, hydrolyze and/or erode. A bioerodible orbiodegradable mass that remains localized and degrades slowly would beparticularly beneficial for treating a wound or diseased or injuredtissue that requires a prolonged treatment regimen.

In other aspects, the micronized compositions described herein can beformulated in any excipient the biological system or entity can tolerateto produce pharmaceutical compositions. Examples of such excipientsinclude, but are not limited to, water, aqueous hyaluronic acid, saline,Ringer's solution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, vegetable oils such as olive oil and sesame oil,triglycerides, propylene glycol, polyethylene glycol, and injectableorganic esters such as ethyl oleate can also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.In certain aspects, the pH can be modified depending upon the mode ofadministration. Additionally, the pharmaceutical compositions caninclude carriers, thickeners, diluents, preservatives, surface activeagents and the like in addition to the compounds described herein.

The pharmaceutical compositions can be prepared using techniques knownin the art. In one aspect, the composition is prepared by admixing amicronized composition described herein with apharmaceutically-acceptable compound and/or carrier. The term “admixing”is defined as mixing the two components together so that there is nochemical reaction or physical interaction. The term “admixing” alsoincludes the chemical reaction or physical interaction between thecompound and the pharmaceutically-acceptable compound.

It will be appreciated that the actual preferred amounts of micronizedcomposition in a specified case will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, and the particular situs and subject being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g. by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate conventional pharmacological protocol. Physicians andformulators, skilled in the art of determining doses of pharmaceuticalcompounds, will have no problems determining dose according to standardrecommendations (Physician's Desk Reference, Barnhart Publishing (1999).

The pharmaceutical compositions described herein can be administered ina number of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. In one aspect, administrationcan be by injection, where the micronized composition is formulated intoa liquid or gel. In other aspects, the micronized composition can beformulated to be applied internally to a subject. In other aspects, themicronized composition can be applied topically (includingophthalmically, vaginally, rectally, intranasally, orally, or directlyto the skin).

In one aspect, the micronized compositions can be formulated as atopical composition applied directly to the skin. Formulations fortopical administration can include, emulsions, creams, aqueoussolutions, oils, ointments, pastes, gels, lotions, milks, foams,suspensions and powders. In one aspect, the topical composition caninclude one or more surfactants and/or emulsifiers.

In some embodiments, localization agents, such as thixotropic agents,phase changing agents, and the like, may include but not limited to,hydrogel, bioerodible, biocompatible polymer, and collagen gels. Thepresence of one or more localization agents in the compositions of thisinvention allows the compositions to have certain viscosity such thatthe compositions are locally retained for a period of time uponadministration or injection. It is within the purview of one of ordinaryskill in the art to determine the suitable viscosity of thecompositions. In some aspects, the compositions have a viscosity betweenabout 5 cP to about 1×10⁸ cP, or about 5 cP to about 1×10⁶ cP, or about5 cP to about 1×10⁵ cP, or about 5 cP to about 1×10⁴ cP, or about 5 cPto about 1×10³ cP, or about 6 cP to about 9500 cP at 25° C.

The hydrogels useful in the compositions of this invention can bechemically and/or physically cross-linked hydrogels. In situ chemicalcross-linking is obtained, e.g., via photo-initiated, redox-initiated orMichael-type addition polymerization that preferably involve covalentbond formation. Physically cross-linked hydrogels self-assemble underexternal stimuli and do not rely on covalent bond formation.Temperature, pH, ion concentration, and hydrophobic interactions arecertain of the external stimuli useful for such self-assembly and forthe immobilization of such hydrogels.

Exemplary polymers suitable for the use in the composition of thepresent invention include polylactides, polyglycolides,poly(caprolactone), polyanhydrides, polyamines, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyphosphoesters, polyorthocarbonates,polyphosphazenes, succinates, poly(malic acid), poly(amino acids),polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose,polyphosphoesters, polysaccharides, chitin, chitosan, hyaluronic acid,and copolymers, terpolymers and mixtures thereof.

Collagens useful in the present invention include Type I, Type III orType I+III collagens, for example, alkaline treatment of insolublecollagen extracted from various animals, or by treating with enzyme suchas pepsin, trypsin, chymotrypsin, papin or pronase. There are noparticular restrictions on the origin of the collagen, and typicallycollagen can be used that is obtained from the skin, bone, cartilage,tendon or organs, etc. of birds or mammals. Since collagen allows theobtaining of a suitable consistency without heating, preparation can bemade easily in the case of gelation. In addition, collagen has a highmolecular weight, it more closely resembles living body tissue, hasconsiderable physiological activity, and therefore promotes healing inthe case of using on a wound, resulting in a further therapeutic effectin combination with the modified placental tissue. Collagen can beflexible after curing and requires only a short time for crosslinking,in other words, requires only a short time for gelation. Collagensolution can also be made by dissolving in a non-toxic solvent respectto the living body, examples of which include water, physiologicalsaline, a buffer such as borate buffer, or an aqueous solutioncontaining a salt such as sodium chloride, sodium bromide and potassiumbromide, or protein, sugar or lipid, etc.

The collagen can also form a gel even in the presence of moisture suchas that in blood or humor, and can demonstrate a high degree ofadhesiveness with respect to living body tissue. Collagen solutions usedin the present invention can be made at various concentrations,neutralized and prepared for injection. In various aspects, collagen at0.2 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mLand 50 mg/mL in solution can be used for injection. Upon injection intoan organ, chilled collagen gels can thermogel as they reach bodytemperature or about 37° C.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. The specification and examples thatfollow should be considered exemplary only.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Example 1—Extraction of Placental Growth Factors

A desired amount of modified placental tissue, which has been previouslycleaned, is extracted in 1M NaCl solution at 4° C. at asolution:modified placental tissue ratio of 10:1 (w/w) for 24 hours.Optionally, the extraction is carried out using a rocker platform underagitation. Following extraction, the supernatant is separated fromresidues by centrifugation. The collected supernatant is then dialyzedagainst water, and subsequently, the solution containing the placentalgrowth factors is lyophilized. Upon administration, the lyophilizedplacental growth factors may be reconstituted in water for injection ata predetermined concentration.

Example 2—Preparation of an Immobilized Composition

Five mL of EpiFix® injectable solution containing a suspension ofmodified placental tissue particles (available from MiMedx Group Inc.,Kennesaw, Ga., USA) is cooled to 5° C. To this solution is addedapproximately 20% w/w of poloxomer PF-127 which is a commerciallyavailable polyoxyethylene-polyoxypropylene triblock copolymer of generalformula E106 P70 E106, with an average molar mass of 13,000 (25,26). Theresulting composition retains its liquid properties at 5° C. but willgelatinate at approximately 20° C.

The cold solution is loaded into a 10 mL syringe and then immediatelyused to inject the solution into a knee joint of a patient exhibiting apartially torn cartilage. Upon injection, the body temperature causes aphase-transfer to a bioerodable gel which will elute the growth factorsduring erosion.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed:
 1. A composition comprising a sufficient amount ofplacental growth factors to treat a diseased or injured organ or bodypart wherein said composition is in the form of a localized mass whenapplied to or proximate to said diseased or injured organ or body part;wherein said composition is free of modified placental tissue particles;wherein said growth factors are extracted from modified placentaltissue; and wherein said modified placental tissue comprises amnionsubstantially free of intermediate layer.
 2. The composition of claim 1,further comprising a localization agent.
 3. The composition of claim 2,wherein the localization agent is a thixotropic agent, or a phasechanging agent.
 4. The composition of claim 3, wherein the thixotropicagent is selected from the group consisting of hyaluronic acid,collagen, thrombin gels, fibrin gels and fibrin glues.
 5. Thecomposition of claim 3, wherein the phase changing agent is a gelforming agent.
 6. The composition of claim 5, wherein the gel formingagent is a copolymer or tripolymer of oxyethylene and oxypropyleneunits.
 7. The composition of claim 2, wherein the localization agent isselected from the group consisting of a hydrogel, a polymer, and acollagen gel.
 8. The composition of claim 1, wherein the compositionforms a localized bioerodible mass when applied to or proximate to saiddiseased or injured organ or body part.
 9. The composition of claim 1,wherein said modified placental tissue further comprises chorion.
 10. Amethod for preparing a composition for localized delivery of placentalgrowth factors, comprising combining an aqueous solution of placentalgrowth factors with a sufficient amount of a localization agent, wherebythe composition is locally retained at the site of delivery uponadministration; wherein said composition is free of modified placentaltissue particles; wherein said growth factors are extracted frommodified placental tissue; and wherein said modified placental tissuecomprises amnion substantially free of intermediate layer.